The present invention relates to a DNA fragment isolated from a retrotransposon and comprising an internal ribosome entry site (IRES). More especially, it relates to expression vectors containing this DNA fragment, and in particular to polycistronic vectors permitting the efficient and stable expression of several genes of interest under the control of the same promoter. The present invention finds advantageous application in the field of gene therapy vectors.
The feasibility of gene therapy applied to man no longer needs to be demonstrated, and this relates to many therapeutic applications such as genetic disorders, infectious diseases and cancer. Many documents of the prior art describe the means of implementing gene therapy, in particular by the use of viral vectors. Generally speaking, the vectors are obtained by deletion of at least a portion of the viral genes which are replaced by the genes of therapeutic interest. Such vectors may be propagated in a complementation line which supplies in trans the deleted viral functions, to generate a viral vector particle which is defective for replication but capable of infecting a host cell. To date, retroviral vectors are among the ones most often used, but vectors originating from adenoviruses, adeno-associated viruses, poxviruses and herpesviruses may also be mentioned. This type of vector, their organization and their mode of infection are widely described in the literature available to a person skilled in the art.
It can be advantageous to have at one's disposal more efficacious gene therapy vectors capable, in particular, of producing several proteins of interest efficiently. However, the presence of several promoters within the same vector very often manifests itself in a reduction or even a loss of expression over time. This is due to a well-known phenomenon of interference between promoter sequences. In this context, the publication of International Application WO93/03143 proposes a solution to this problem which consists in employing an internal ribosome entry site (IRES). It describes a dicistonic retroviral vector for the expression of two genes of interest placed under the control of the same promoter. The presence of a picornavirus IRES site between these genes permits the production of the expression product originating from the second gene of interest by internal initiation of the translation of the dicistronic MRNA.
Normally, the entry of ribosomes into messenger RNA takes place via the cap located at the 5' end of all eukaryotic mRNAs. However, there are exceptions to this universal rule. The absence of a cap in some viral mRNAs suggests the existence of alternative structures permitting the entry of ribosomes at an internal site of these RNAs. To date, a number of these structures, designated IRES on account of their function, have been identified in the 5' noncoding region of uncapped viral mRNAs, such as that, in particular, of picornaviruses such as the poliomyelitis virus (Pelletier et al., 1988, Mol. Cell. Biol., 8, 1103-1112) and the EMCV virus (encephalo-myocarditis virus (Jang et al., J. Virol., 1988, 62, 2636-2643).